Project Summary/Abstract How proteins fold, that is attain their three-dimensional structure, is a fundamental biological process with important implications for human health. Misfolded proteins are often toxic, as illustrated by the number of neurodegenerative diseases referred to as protein folding diseases. Molecular chaperones play vital roles in remodeling protein structure -- assisting de novo protein folding, preventing protein aggregation and disassembling protein complexes. Hsp70-based machineries, having J-proteins as obligate components, are amongst the most highly conserved molecular chaperone systems. J-proteins are a very diverse set of proteins, having only the 70 amino acid J-domain in common. All J-proteins share the ability to stimulate the ATPase activity of their partner Hsp70s, allowing them to capture client proteins. But it is their functional diversity that enables them to orchestrate Hsp70s capacity to participate in a wide array of complex and diverse biological functions. This proposal focuses on understanding the basis of the specificity of J-proteins function. Two J-proteins of the yeast cytosol have been chosen for in depth analysis: Sis1 and Zuo1. This choice is based on their critical importance and their high degree of sequence conservation. Their human homologs are able to substitute for the yeast proteins, thus the outcome of this work will serve as a paradigm for understanding J-protein function in other organisms. To understand the specificity of Sis1, its function in the propagation of yeast prions will be exploited, as it is specifically required for fragmentation of prion complexes. Thus, results will also yield important information about the biogenesis and propagation of these self-replicating amyloid protein aggregates. Zuo1 is a highly conserved ribosome-associated chaperone that facilitates interaction of Hsp70 with nascent polypeptides as they exit the ribosome. Ribosome-associated chaperones serve as a link between protein synthesis and protein folding and are thus a key to the cells production of functional proteins. In addition, we will investigate roles of molecular chaperones in the nucleus, focusing on novel regulatory functions independent of and separable from chaperone activity.